The human leukocyte antigen complex, also known as the major histocompatibility complex, spans approximately 3.5 million base pairs on the short arm of chromosome 6. It is divisible into 3 separate regions, which contain the class I, the class II and the class III genes, respectively. There are 20 genes in the class I region, including the classical MHC molecules designated HLA-A, HLA-B and HLA-C. In addition are the nonclassical class I genes: HLA-E, HLA-F, HLA-G, HLA-H, HLA-J, HLA-X, and MIC. The class II region contains the HLA-DP, HLA-DQ and HLA-DR loci, which encode the α and β chains of the classical class II MHC molecules designated HLA-DR, DP and DQ. Nonclassical genes designated DM, DN and DO have also been identified within class II. The class III region contains a heterogeneous collection of more than 36 genes. The loci constituting the MHC are highly polymorphic. Several hundred different allelic variants of class I and class II MHC molecules have been identified in humans.
The specific protein sequences of the highly polymorphic HLA locus play a major role in determining histocompatibility of transplants, as well as important insight into susceptibility of a number of immune related disorders, including celiac disease, rheumatoid arthritis, insulin dependent diabetes mellitus, multiple sclerosis and the like. Matching of donor and recipient HLA-DR and DQ alleles prior to allogeneic transplantation has a particularly important influence on allograft survival. Therefore, HLA matching is universally required as a clinical prerequisite for renal and bone marrow transplantation as well as cord blood applications.
Conventional matching has been performed by serological and cellular typing. For example, in a microcytotoxicity test, white blood cells from the potential donor and recipient are distributed in a microtiter plate and monoclonal antibodies specific for class I and class II MHC alleles are added to different wells. Thereafter, complement is added to the wells and cytotoxicity is assessed by uptake or exclusion to various dyes by the cells. However, serological typing is frequently problematic, due to the availability and crossreactivity of alloantisera and because live cells are required. A high degree of error and variability is also inherent in serological typing. Therefore, DNA typing is becoming more widely used as an adjunct, or alternative, to serological tests.
In some methods, PCR amplified products are hybridized with sequence-specific oligonucleotide probes (PCR-SSO) to distinguish between HLA alleles. This method requires a PCR product of the HLA locus of interest be produced and then dotted onto nitrocellulose membranes or strips. Then each membrane is hybridized with a sequence specific probe, washed, and then analyzed by exposure to x-ray film or by colorimetric assay depending on the method of detection. Hybridization and detection methods for PCR-SSO typing include the use of non-radioactive labeled probes, microplate formats, etc., and automated large scale HLA class II typing.
More recently, a molecular typing method using sequence specific primer amplification (PCR-SSP) has been described. In PCR-SSP, allelic sequence specific primers amplify only the complementary template allele, allowing genetic variability to be detected with a high degree of resolution. This method allows determination of HLA type simply by whether or not amplification products are present or absent following PCR. In PCR-SSP, detection of the amplification products may be done by agarose gel electrophoresis.
Currently, direct DNA sequencing or “sequence based typing” (SBT) provides the highest resolution to discriminate these alleles at the nucleotide level, where minor differences in sequence have great impact on the phenotype of the HLA genes. However, HLA genes span between 5 Kb to 15 Kb interspersed between introns and exons in human genome. All current DNA sequencing approaches target one or a few of disjoined exons in the genomic DNA and not the processed RNA from these genes because of the inherent difficulty of handling RNA in patient samples. Since each individual is diploid, it is important to characterize the unique sequence from each gene to understand how these changes are reflected at the protein level. Without linkage information between those exons, the fragmental information from individual exons generates incomplete data and is not sufficient for definitive haplotype determination.
Improved methods of HLA typing are of great interest for research and clinical applications.